PROJECT SUMMARY As a ubiquitous HSP90 paralog in the endoplasmic reticulum, grp94 plays important roles in protein quality control in the secretory pathway by participating in both the unfolded protein response and the ER-associated protein degradation pathway. It is over-expressed in cancers, with higher levels conferring poorer prognosis, but the underlying relationship between expression levels and pathology is unclear. Historically, the biology of grp94 has been studied in the context of the host's immune response to cancer, but not in the context of cancer biology per se. For the past decade, our laboratory has focused on determining the intrinsic properties of grp94 via genetic approaches. Using the cre-loxP system, we generated a conditional grp94 knockout (KO) mouse model. Functional analyses of various cell type-specific grp94 null mice have led to the discovery that grp94 is an obligate chaperone controlling several key pathways for cell growth, migration and differentiation, including integrins, Toll-like receptors (TLRs), Wnt co-receptor LRP6, and the cell surface TGF?-docking molecule GARP. Thus, grp94 has emerged as a strategically important target for cancer therapy. This project will focus on the breast cancer biology of the grp94-GARP-TGF? axis. Our overarching hypothesis is that grp94 is a key molecular chaperone in regulating the expression of cell surface TGF? through GARP as well as its activation process via integrins. Strong preliminary data support this hypothesis. We have obtained both in vitro and in vivo evidence that grp94 controls cell surface expression of GARP as well as integrins. GARP is required for docking latent TGF? to cell surface, whereas integrins contribute to latent TGF? activation. The cancer relevance of grp94-GARP-TGF? axis in cancer has been established by new preliminary data including extensive immunohistochemistry of tumor/tissue microarrays of multiple primary human cancers, demonstrating the correlation between aberrant expression of GARP protein in cancers and worse survival. The impact of GARP overexpression in promoting breast cancer and immune tolerance also was observed. Thus, we are poised to study both the mechanism and the biological impact of the grp94-GARP-TGF? axis in cancer biology. Our first aim will establish GARP as a novel grp94 client protein, determine the roles of grp94 in regulating surface TGF? biogenesis and activation, and uncover the detailed mechanism by which grp94 regulates GARP maturation and trafficking. Our second aim will pinpoint the biological significance of grp94- GARP expression in de novo oncogenesis using several novel mouse models that allow precise control of GARP expression genetically. Using a panel of unique GARP monoclonal antibodies, Aim 3 will determine the clinical significance of grp94-GARP expression in breast cancer and address if GARP can serve as a novel therapeutic target. This project will advance the field of grp94 biology in cancer by providing timely answers to many outstanding questions and by paving the way to eventually unlock the regulatory circuit of the grp94- GARP-TGF? pathway for fundamental understanding, prognosis and treatment of human cancers.